Hemagglutinin (HA) is the major surface membrane glycoprotein responsible for the binding of influenza virus to sialic-acid containing receptors in target cells. Fusion of viral and endosomal membranes is triggered by an acid-induced conformational change of HA, which takes place in the low pH of the endosomes. As a result, the viral transcriptase complex is transferred to the cell and viral replication is initiated. HA is a trimeric integral membrane glycoprotein (MW 220,000) comprised of an ectodomain of identical subunits, each of which contains two polypeptides, HA1 and HA2, linked by a disulfide bond. While HA1 is the receptor-binding subunit, HA2 mediates fusion through a low pH-dependent conformational change that leads to exposure of the highly hydrophobic N-terminus, the fusion peptide. A manuscript on the following results has been provisionally accepted by the Journal of Biochemistry pending revision. The conformational stability of influenza virus (strain X31) hemagglutinin (HA) has been investigated using differential scanning calorimetry (DSC), analytical ultracentrifugation, Trp fluorescence, and circular dichroism (CD) as a function of pH since viral fusion is triggered by acidification. HA sediments as a spherical complex (rosette) comprised of 5-6 trimers (31-35 S) over the pH range of 7.4 to 5.4. A single DSC endotherm of HA at pH 7.4 in the absence (and presence of 1% octyl-beta-glucoside) shows three domains with a transition temperature of 66.0 +/- 0.2 (65.0) C and overall enthalpy change of 800 +/- 80 kcal/(mol trimer) even though HA rosettes are dissociated to trimers (10.3 S) in detergent. Intermolecular interactions among trimers in rosettes at pH 7.4 therefore contribute at most ~1 K stabilization. As the pH is decreased from pH 7.4 to 5.4, transition temperatures decrease from 66 to 45 C and unfolding enthalpies decrease from 880 to 260 kcal/(mol trimer) with only one endotherm observed at each pH. Corresponding ratios of calorimetric to van?t Hoff enthalpies decrease from 3.0 (at pH 7.4) to -1.3 (at pH 5.4). Overall secondary and tertiary structures are maintained throughout this pH range (as shown by far- and near-UV CD spectra), and the changes in HA structure upon acidification are well documented. Disruption of HA1/HA1 contacts in a trimer account for the observed proton-induced increase in surface hydrophobicity and reduction of intrinsic tryptophan fluorescence below pH 6.0, as well as for the large decreases in transition temperatures for thermal unfolding. During thermal unfolding of protonated, dissociated HA1 regions, interactions with other partially unfolded HA1 domains within the trimer apparently decrease cooperativity ratios (calorimetric to van?t Hoff enthalpies) expressed per trimer. Optimal temperatures for viral fusion (e.g., 37 C at pH 5.4) are lower than transition temperatures of HA at different acidic pH values, which indicates that dissociated HA1 distal domains must be folded for competent viral fusion.